Multiplexed fluorescence readout using time responses of color coded signals for biomolecular detection

Fluorescence readout is an important technique for detecting biomolecules. In this paper, we present a multiplexed fluorescence readout method using time varied fluorescence signals. To generate the fluorescence signals, coded strands and a set of universal molecular beacons are introduced. Each coded strand represents the existence of an assigned target molecule. The coded strands have coded sequences to generate temporary fluorescence signals through binding to the molecular beacons. The signal generating processes are modeled based on the reaction kinetics between the coded strands and molecular beacons. The model is used to decode the detected fluorescence signals using maximum likelihood estimation. Multiplexed fluorescence readout was experimentally demonstrated with three molecular beacons. Numerical analysis showed that the readout accuracy was enhanced by the use of time-varied fluorescence signals.